Format

Send to

Choose Destination
Mol Ther Methods Clin Dev. 2019 Nov 26;17:107-121. doi: 10.1016/j.omtm.2019.11.014. eCollection 2020 Jun 12.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

Author information

1
Grousbeck Gene Therapy Center, Schepens Eye Research Institute, Mass Eye and Ear, Boston, MA 02114, USA.
2
INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.
3
Ocular Genomics Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114, USA.
4
The Broad Institute of Harvard and MIT, Cambridge, MA, USA.
5
Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA.

Abstract

Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multiplexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombinant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.

KEYWORDS:

aav; adeno-associated virus; barcode; capsid; capsid mosaics; cross-packaging; library; mosaic; viral genome

Supplemental Content

Full text links

Icon for Elsevier Science Icon for PubMed Central
Loading ...
Support Center